Global Predictions and Tests of Erythroid Regulation

红细胞调节的全球预测和测试

• 批准号: 6847425
• 负责人: ROSS C HARDISON
• 金额: $ 45.23万
• 依托单位: PENNSYLVANIA STATE UNIVERSITY, THE
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-02-01 至 2009-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6847425
• 关键词: DNA binding protein; binding sites; chromatin immunoprecipitation; computer assisted sequence analysis; erythroid stem cell; erythroleukemia; gene expression; gene induction /repression; genetic regulation; genetic regulatory element; genome; green fluorescent proteins; molecular biology information system; nucleic acid hybridization; nucleic acid sequence; polymerase chain reaction; transcription factor

DESCRIPTION (provided by applicant): New technologies to better define the structure and expression of mammalian genomes offer exciting opportunities for understanding the genetic control of tissue development. Recent studies in our (two separate) laboratories used cell-based, molecular and bioinformatic approaches to generate potentially important insights into the structure, function and expression of erythroid genes. Now we aim to combine our preliminary data and expertise to conduct a genome-wide search for erythroid regulatory modules, i.e. the DNA sequences and proteins needed for erythroid regulation. This project will also serve as a testing ground for new genome-wide computational predictions of regulatory elements. We propose to define cohorts of co-regulated genes in induced murine erythroleukemia (MEL) cells and in Gata-1-null erythroid precursors (G1E cells) induced to differentiate by restoration of GATA-1 function. We will search for likely regulatory regions in noncoding genomic sequences encompassing these genes by using programs that analyze whole-genome human-mouse alignments to predict functional and regulatory sequences, plus searches for clusters of erythroid transcription factor binding sites. Strong candidates for regulatory sequences will be tested in erythroid expression cassettes that will be integrated into a targeted location in MEL cell and G1E cell chromosomes. We will measure their effects on expression using reporters such as green fluorescent protein and luciferase. After analyzing these results for features of the sequences and alignments that best correlate with demonstrable enhancer activity, we will incorporate those features into our programs that seek to discriminate regulatory sequences from other sequences. Reiterative cycles of analysis and experimental validation will lead to more effective predictions of erythroid regulatory sequences. Our comprehensive analysis of cis regulatory elements should lead to improved understanding of how gene expression is controlled and coordinated during erythroid maturation, a topic of relevance to the biology of hematopoiesis and to therapeutic strategies for hemoglobinopathies. More broadly, the experimental and computational approaches generated by our work should provide valuable tools for analyzing the control of gene expression in other tissues during normal development and various pathological states.

描述（由申请人提供）： 新技术，以更好地确定哺乳动物基因组的结构和表达提供了令人兴奋的机会，了解组织发育的遗传控制。我们（两个独立的）实验室最近的研究使用了基于细胞的分子和生物信息学方法，对红细胞基因的结构、功能和表达产生了潜在的重要见解。现在，我们的目标是联合收割机结合我们的初步数据和专业知识，进行全基因组搜索红细胞调控模块，即红细胞调控所需的DNA序列和蛋白质。该项目还将作为新的全基因组计算预测调控元件的试验场。我们建议在诱导的小鼠红白血病（MEL）细胞和加塔-1-null红系前体细胞（G1 E细胞）中定义共调节基因的队列，通过恢复加塔-1功能诱导分化。我们将通过使用分析全基因组人-鼠比对的程序来预测功能和调控序列，再加上搜索红细胞转录因子结合位点簇，在包含这些基因的非编码基因组序列中搜索可能的调控区域。将在红系细胞表达盒中检测调控序列的强候选物，所述红系细胞表达盒将整合到MEL细胞和G1 E细胞染色体的靶向位置中。我们将使用诸如绿色荧光蛋白和荧光素酶的报告物来测量它们对表达的影响。在分析这些结果的特征的序列和比对，最好与可证明的增强子活性，我们将这些功能纳入我们的计划，寻求从其他序列的监管序列的区别。重复的分析和实验验证循环将导致更有效的预测红细胞调控序列。我们的顺式调控元件的全面分析，应导致更好地了解基因表达是如何控制和协调红细胞成熟，造血生物学和血红蛋白病的治疗策略的相关主题。更广泛地说，我们的工作产生的实验和计算方法应该为分析正常发育和各种病理状态期间其他组织中基因表达的控制提供有价值的工具。